Toll-like receptor 2 and 4 mediated effects on human platelet function

In addition to their specific role in haemostasis and thrombus formation, platelets are also involved in immune response mechanisms. Toll like receptors (TLRs) represent a component of this innate defense system, and as so-called pattern recognition receptors (PRRs), are jointly responsible for the recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). This work examines the functional mechanisms associated with TLR2 and TLR4 and their interplay in anucleated platelets. The elucidation of these mechanisms contributes to a better understanding of TLR-mediated physiology and pathophysiology, possibly relevant for transfusion reactions or infectious diseases. Initially, it was demonstrated that the method of platelet preparation, either as platelet rich plasma (PRP) or as washed platelets (WP), has a significant influence on TLR mediated effects. Aggregation was only observed in washed platelets (WP) stimulated with the Toll like receptor (TLR) 2 agonist Pam3CSK4 and the TLR4 agonist lipopolysaccharide (LPS from E. coli) at a concentration of 15 µg/ml. LPS alone resulted in stable aggregation when combined with a low thrombin dose of 0.025 U/ml. The release of physiologically relevant chemokines was observed at a concentration of 15 µg/ml for Pam3CSK4 and LPS. Induced concentrations were found to be higher with the TLR2 agonist and to increase over time. Significantly higher concentrations were achieved with classical platelet activators in potent concentrations, 10 µM TRAP-6 (thrombin receptor activating peptide 6) and 0.5 U/ml thrombin, indicating submaximal effects of TLR stimulation on secretion. Western blot analysis was employed to analyse the involved signalling pathways. The results confirmed that TLR2 induced effects have a higher potential than TLR4 induced effects with respect to proteins of the classical activating pathways. No significant differences were observed for inhibitory signalling pathways after stimulation of the two receptors. Moreover, the NFκB signalling pathway, which is crucial in nucleated cells, was examined with an opposing outcome. LPS-mediated stimulation resulted in a decrease in Akt/PKB phosphorylation, which was accompanied by a reduction in protein kinase 2A (PP2A) phosphorylation. These findings were further validated by adhesion studies using immunofluorescence under static conditions. Given that PP2A is known to be associated with cytoskeletal rearrangements and platelet spreading, it was demonstrated that a larger surface area was covered by adherent platelets upon stimulation with LPS compared to Pam3CSK4. In conclusion, this work can be summarized by the fact that the choice of platelet preparation method has a decisive influence on the results to be obtained. This can explain discrepant results in previous studies. The plasma components present in PRP can exert an inhibitory effect at the level of Toll like receptors (TLRs) or contribute to the neutralization of chemokines. This release, which can be modulated by plasma, is of particular relevance for transfusion medicine with regard to platelet storage environments. Moreover, despite their initial similarity, the stimulation of TLR2 and TLR4 lead to different effector mechanisms in platelets. The stimulation of TLR2 via Pam3CSK4 is associated with classical platelet activation and aggregation compared to stimulation of TLR4 via LPS. However, the latter exerts specific influence on platelet signalling via Akt/PKB and PP2A.